Compositions for mitigating hydrogen sulfide contamination using a recombinant protein with an affinity tag fused to a hydrogen sulfide scavenging enzyme

ABSTRACT

In some embodiments, the present invention provides a recombinant protein comprising an affinity tag configured to attach the recombinant protein to a silicate surface, fused to a hydrogen sulfide scavenging enzyme.

RELATED APPLICATIONS

This application claims the priority of U.S. provisional application U.S. Patent Appln. No. 62/357,025; filed Jun. 30, 2016; entitled “COMPOSITIONS AND METHODS FOR MITIGATING HYDROGEN SULFIDE CONTAMINATION,” which is incorporated herein by reference in its entirety for all purposes.

TECHNICAL FIELD

In some embodiments, the present invention is related to compositions and methods for attaching proteins to silicate surfaces. In particular, in some embodiments, the present invention is related to compositions and methods for mitigating hydrogen sulfide and/or mercaptan contamination of a liquid within a reservoir using an enzyme attached to the silicate surface of a reservoir.

BACKGROUND

Hydrogen sulfide and mercaptans are present in underground water removed with crude oil, in crude oil itself, in natural gases and in gases associated with underground water and crude oil. Hydrogen sulfide and mercaptans are characterized by highly noxious odors and typically are highly corrosive. Uncontrolled emissions of hydrogen sulfide give rise to severe health hazards. The presence of hydrogen sulfide and mercaptans is further objectionable because they often react with desirable hydrocarbons as well as fuel system components.

SUMMARY OF INVENTION

In one embodiment, the present invention provides a recombinant protein comprising an affinity tag configured to attach the recombinant protein to a silicate surface, fused to a hydrogen sulfide scavenging enzyme.

In one embodiment, the affinity tag configured to attach the recombinant protein to a silicate surface is nonarginine (i.e., non-, meaning nine, arginine residues: RRRRRRRRR).

In one embodiment, the affinity tag configured to attach the recombinant protein to a silicate surface is the 30 kDa L2 protein from the E. Coli 50S ribosomal subunit.

In one embodiment, the hydrogen sulfide scavenging enzyme is sulfide quinone reductase.

In one embodiment, the sulfide quinone reductase is derived from Acidophilus ferroxidans.

In one embodiment, the recombinant protein comprising an affinity tag configured to attach the recombinant protein to a silicate surface, fused to a hydrogen sulfide scavenging enzyme comprises the protein having the amino acid sequence set forth in SEQ ID NO: 1. The recombinant protein is referred to herein as “SQR-R₉”.

In one embodiment, the recombinant protein comprises the protein having the amino acid sequence set forth in SEQ ID NO: 1.

In one embodiment, the recombinant protein comprising an affinity tag configured to attach the recombinant protein to a silicate surface, fused to a hydrogen sulfide scavenging enzyme comprises the protein having the amino acid sequence set forth in SEQ ID NO: 2. The recombinant protein is referred to herein as “SQR-L2”.

In one embodiment, the recombinant protein comprises the protein having the amino acid sequence set forth in SEQ ID NO: 2.

In one embodiment, the affinity tag is the peptide having the amino acid sequence set forth in SEQ ID NO: 3.

In one embodiment, the affinity tag is the peptide having the amino acid sequence set forth in SEQ ID NO: 4.

In one embodiment, the present invention provides a method, wherein the method scavenges hydrogen sulfide and/or mercaptans from a liquid within a reservoir defined by a solid silicate surface, the method comprising contacting the solid silicate surface with a catalytically effective amount of the recombinant protein according to some embodiments of the present invention, wherein the recombinant protein catalyzes the oxidation of the hydrogen sulfide and/or mercaptans to a sulfur containing oxidation product.

In one embodiment, the present invention provides a method, wherein the method inhibits the production of hydrogen sulfide and/or mercaptans from a liquid within a reservoir defined by a solid silicate surface, the method comprising contacting the solid silicate surface with a catalytically effective amount of the recombinant protein according to some embodiments of the present invention, wherein the recombinant protein catalyzes the oxidation of the hydrogen sulfide and/or mercaptans to a sulfur containing oxidation product.

In one embodiment, the liquid is sour well water.

In one embodiment, the liquid is sea water.

In one embodiment, the liquid is brine.

BRIEF DESCRIPTION OF DRAWINGS

The present invention will be further explained with reference to the attached drawings, wherein like structures are referred to by like numerals throughout the several views. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the present invention. Further, some features may be exaggerated to show details of particular components.

FIG. 1 shows a recombinant protein according to some embodiments of the present invention immobilized onto sand.

FIG. 2 shows a recombinant protein according to some embodiments of the present invention immobilized onto sandstone wafers.

FIG. 3 shows a recombinant protein according to some embodiments of the present invention immobilized onto sandstone cores.

Among those benefits and improvements that have been disclosed, other objects and advantages of this invention will become apparent from the following description taken in conjunction with the accompanying figures. Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the invention that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the invention which are intended to be illustrative, and not restrictive.

DETAILED DESCRIPTION

The present invention will be further explained with reference to the attached drawings, wherein like structures are referred to by like numerals throughout the several views. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the present invention. Further, some features may be exaggerated to show details of particular components.

The figures constitute a part of this specification and include illustrative embodiments of the present invention and illustrate various objects and features thereof. Further, the figures are not necessarily to scale, some features may be exaggerated to show details of particular components. In addition, any measurements, specifications and the like shown in the figures are intended to be illustrative, and not restrictive. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrases “in one embodiment” and “in some embodiments” as used herein do not necessarily refer to the same embodiment(s), though it may. Furthermore, the phrases “in another embodiment” and “in some other embodiments” as used herein do not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

In addition, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. The meaning of “in” includes “in” and “on.”

As used herein, the term “mercaptan” shall include alkyl mercaptans and thiols of the formula R—SH where R is an unsubstituted or substituted alkyl, thiol carboxylic acids and dithio acids.

As used herein, the term “aqueous substrate” shall refer to any “sour” aqueous substrate, including waste water streams in transit to or from municipal waste water treatment facilities, tanning facilities, and the like.

The term “hydrocarbon substrate” is meant to include unrefined and refined hydrocarbon products, including natural gas, derived from petroleum or from the liquefaction of coal, both of which contain hydrogen sulfide or other sulfur-containing compounds. Thus, particularly for petroleum-based fuels, the term “hydrocarbon substrate” includes, but is not limited to, wellhead condensate as well as crude oil which may be contained in storage facilities at the producing field. “Hydrocarbon substrate” also includes the same materials transported from those facilities by barges, pipelines, tankers, or trucks to refinery storage tanks, or, alternately, transported directly from the producing facilities through pipelines to the refinery storage tanks. The term “hydrocarbon substrate” also includes refined products, interim and final, produced in a refinery, including distillates such as gasoline, distillate fuels, oils, and residual fuels and to vapors produced by the foregoing materials.

Hydrogen Sulfide and/or Mercaptan Scavenging Enzymes According to Some Embodiments of the Present Invention

In some embodiments, the present invention provides a recombinant protein comprising an affinity tag configured to attach the recombinant protein to a silicate surface, fused to a hydrogen sulfide scavenging enzyme.

In some embodiments, the hydrogen sulfide scavenging enzyme is a sulfide quinone reductase (SQR) enzyme.

Without intending to be limited to any particular theory, the sulfide quinone reductase (SQR) enzyme prevents the formation of hydrogen sulfide and mercaptans in a liquid within a reservoir. The formation of hydrogen sulfide and/or mercaptans can contribute to corrosion of the materials of the reservoir, and vessels used to transport, store, and/or manufacture the liquid.

In some embodiments, the sulfide quinone reductase (SQR) enzyme may originate from various organisms.

In some embodiments, the SQR enzyme is derived from Acidophilus ferroxidans.

In some embodiments, the SQR enzyme may be derived from a gram negative, acidophilic and thermophilic bacterium, such as Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), or any combination thereof.

In some embodiments, a nucleotide sequence encoding the SQR enzyme may be derived from a gram negative, acidophilic and thermophilic bacterium, such as Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula, using polymerase chain reaction (PCR) amplification.

In some embodiments, the SQR enzyme is the SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1, which is incorporated herein by reference in its entirety.

In some embodiments, the sulfide quinone reductase (SQR) enzyme is isolated according to the methods described in U.S. Patent Application Publication No. 20160039697 A1, which is incorporated herein by reference in its entirety.

In some embodiments, the hydrogen sulfide scavenging enzyme is cysteine synthase.

In some embodiments, the cysteine synthase is derived from Acidithiobacillus caldus SM-1. Alternatively, in some embodiments, the cysteine synthase is derived from Aeropyrum pernix.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 80% homologous to a SQR derived from, e.g., but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 85% homologous to a SQR derived from, e.g., but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 90% homologous to a SQR derived from, e.g., but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 95% homologous to a SQR derived from, e.g., but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 96% homologous to a SQR derived from, e.g., but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 97% homologous to a SQR derived from, e.g., but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 98% homologous to a SQR derived from, e.g., but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 99% homologous to a SQR derived from, e.g., but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 80% and 99% homologous to a SQR derived from, but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 85% and 99% homologous to a SQR derived from, but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 90% and 99% homologous to a SQR derived from, but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 99% homologous to a SQR derived from, but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 96% and 99% homologous to a SQR derived from, but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 97% and 99% homologous to a SQR derived from, but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 98% and 99% homologous to a SQR derived from, but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 98% homologous to a SQR derived from, but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 97% homologous to a SQR derived from, but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 96% homologous to a SQR derived from, but not limited to, Acidophilus ferroxidans, Acidithiobacillus albertensis (SEQ ID NO: 8), Thiohalospira halophila DSM 15071 (SEQ ID NO: 9), endosymbiont of Riftia pachyptila (vent Ph05) (SEQ ID NO: 10), Acidovorax soli (SEQ ID NO: 11), Thiothrix caldifontis (SEQ ID NO: 12), Acidithobacillus ferroxidans, Metallospora cuprina or Metallospora sedula.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 80% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 85% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 90% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 95% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 80% and 99% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 85% and 99% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 90% and 99% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 99% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 96% and 99% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 97% and 99% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 98% and 99% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 98% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 97% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 96% homologous to a SQR enzyme disclosed in U.S. Patent Application Publication No. 20160039697 A1.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 80% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 85% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 90% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 95% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 96% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 97% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 98% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme at least 99% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 80% and 99% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 85% and 99% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 90% and 99% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 99% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 96% and 99% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 97% and 99% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 98% and 99% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix.

In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 98% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 97% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix. In some embodiments, the hydrogen sulfide scavenging enzyme is an enzyme between 95% and 96% homologous to a cysteine synthase derived from, e.g., but not limited to, Acidithiobacillus caldus SM-1 or Aeropyrum pernix.

Affinity Tags Suitable for Use in the Hydrogen Sulfide and/or Mercaptan Scavenging Enzymes According to Some Embodiments of the Present Invention

In some embodiments, the present invention provides a recombinant protein comprising an affinity tag configured to attach the recombinant protein to a silicate surface, fused to a hydrogen sulfide scavenging enzyme.

Without intending to be limited to any particular theory, in some embodiments, the affinity tag has an affinity for silicates. In some embodiments the silicate is the silicate surface of a reservoir, pipe, or storage vessel containing a liquid, thereby enabling a protein, such as, the hydrogen sulfide scavenging enzyme, that contains the affinity tag to bind to the silicate surface, such that the protein remains fixed to the silicate surface.

In some embodiments, the affinity tag enables the recombinant protein according to some embodiments of the present invention to remain attached to the silicate surface during any natural or induced flux of the liquid through the reservoir, pipe, or storage vessel.

In some embodiments, the affinity tag is fused to the hydrogen sulfide scavenging enzyme such that the affinity tag does not interfere with the catalytic activity of the hydrogen sulfide scavenging enzyme. Accordingly, in some embodiments, the affinity tag may be attached to the hydrogen sulfide scavenging enzyme via linker.

Factors that may influence the catalytic activity of the hydrogen sulfide scavenging enzyme include, but are not limited to, the relative size of the affinity tag compared to the hydrogen sulfide scavenging enzyme, the distance between the affinity tag and the hydrogen sulfide scavenging enzyme, the relative orientation of the affinity tag and the hydrogen sulfide scavenging enzyme (i.e., if the affinity tag is fused to either the N- or C-terminus of the hydrogen sulfide scavenging enzyme), and the like.

In some embodiments, the affinity tag is fused to the N-terminus of the hydrogen sulfide scavenging enzyme. Alternatively, in some embodiments, the affinity tag is fused to the C-terminus of the hydrogen sulfide scavenging enzyme.

In some embodiments, the affinity tag is incorporated into the hydrogen sulfide scavenging enzyme. In some embodiments, the incorporation orients the active site of the hydrogen sulfide scavenging enzyme away from the silicate surface, thereby reducing stearic hinderance.

In some embodiments, the affinity tag configured to attach the recombinant protein to a silicate surface is nonarginine.

In some embodiments, the affinity tag configured to attach the recombinant protein to a silicate surface is the 30 kDa L2 protein from the E. Coli 50S ribosomal subunit.

In some embodiments, the affinity tag is the peptide having the amino acid sequence set forth in SEQ ID NO: 3.

In some embodiments, the affinity tag is the peptide having the amino acid sequence set forth in SEQ ID NO: 4.

In some embodiments, the affinity tag is a polyhistidine. In some embodiments, the polyhistidine tag comprises at least 6 histidine (HIS) residues. In some embodiments, the polyhistidine tag binds to a metal, e.g., but not limited to, a metal comprising nickel.

In some embodiments, the recombinant protein comprising an affinity tag configured to attach the recombinant protein to a silicate surface, fused to a hydrogen sulfide scavenging enzyme comprises the protein having the amino acid sequence set forth in SEQ ID NO: 1.

In some embodiments, the recombinant protein comprising an affinity tag configured to attach the recombinant protein to a silicate surface, fused to a hydrogen sulfide scavenging enzyme comprises the protein having the amino acid sequence set forth in SEQ ID NO: 2.

In some embodiments, the recombinant protein comprising an affinity tag configured to attach the recombinant protein to a silicate surface, fused to a hydrogen sulfide scavenging enzyme is expressed in a bacterial expression system, isolated, purified, and added to the solid silicate surface in a catalytically effective amount. Methods to generate, express, and/or purify recombinant protein using bacterial expression systems are readily selected by one of ordinary skill in the art.

Methods of Scavenging Hydrogen Sulfide and/or Mercaptans According to Some Embodiments of the Present Invention

In some embodiments, the present invention provides a method, wherein the method scavenges hydrogen sulfide and/or mercaptans from a liquid within a reservoir defined by a solid silicate surface, the method comprising contacting the solid silicate surface with a catalytically effective amount of the recombinant protein according to some embodiments of the present invention, wherein the recombinant protein catalyzes the oxidation of the hydrogen sulfide and/or mercaptans to a sulfur containing oxidation product.

In some embodiments, the present invention provides a method, wherein the method inhibits the production of hydrogen sulfide and/or mercaptans from a liquid within a reservoir defined by a solid silicate surface, the method comprising contacting the solid silicate surface with a catalytically effective amount of the recombinant protein according to some embodiments of the present invention, wherein the recombinant protein catalyzes the oxidation of the hydrogen sulfide and/or mercaptans to a sulfur containing oxidation product.

Generally, for industrial or commercial use, the recombinant protein according to some embodiments of the present invention may be contacted with a stream containing the hydrogen sulfide or mercaptans for removal, and allowed to attach to the silicate surface enclosing the stream. Contact can occur in a variety of containers, such as a process or transport line, a separate stirred or non-stirred container or other vessels such as scrubbers or strippers. Further, the recombinant protein according to some embodiments of the present invention may be added via surface or downhole equipment or at any time in the process stream in recovering crude oil, and allowed to attach to the silicate surface, so as to remove the noxious quality and corrosive nature of the hydrogen sulfide and mercaptans in the processing system.

The methods according to some embodiments of the present invention have applicability in the removal of hydrogen sulfide and mercaptans of the formula R—SH wherein R is an alkyl group having from 1 to 40 carbon atoms, alternatively from 1 to 20 carbon atoms, alternatively from 1 to 6 carbon. Without intending to be limited by any particular theory, such mercaptans have noxious odors and are corrosive.

The methods defined herein are applicable to a wide variety of fluid streams, including liquefied petroleum gas as well as crude oil and petroleum residual fuel, heating oil, etc. In addition, the method are applicable to gaseous hydrocarbon streams.

In some embodiments, the liquid is water.

In some embodiments, the liquid is sour well water.

In some embodiments, the liquid is a salt water.

In some embodiments, the liquid is sea water.

In some embodiments, the liquid is brine.

In some embodiments, the liquid is not a hydrophobic solvent, e.g., but not limited to, an oil.

In some embodiments, the catalytically effective amount of SQR enzyme is an amount sufficient to effectuate the desired result over a sustained period of time and thus is dependent on the amount of the hydrogen sulfide and/or mercaptan in the medium being treated. In general, the amount of the SQR enzyme added to the medium is at least an effective scavenging amount, for example, from about 0.05 ppm to about 2,000 ppm or more, alternatively from about 20 to about 1,200 ppm, and alternatively from about 100 to about 400 ppm of hydrogen sulfide and/or mercaptan.

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

EXAMPLES Example 1: Binding of the Recombinant Proteins According to Some Embodiments of the Present Invention to Silicate Substrates

The sequence for enhanced GFP (eGFP) was used as a model system. The sequence was obtained from the National Center for Biotechnology Information and developed in silico with an endopeptidase cleavage site, a five amino acid linker and a nine arginine repeat on the C-terminal end of the protein. Nde1 and BamH1 restriction endonuclease sites were cloned onto the 5′ and 3′ ends of the gene, respectively. The sequence was codon optimized, using the methods described in U.S. Pat. No. 8,326,547, which are incorporated by reference herein in their entireties. The sequences was then submitted to Genscript for production and cloning into a pET-expression vector from Agilent. Plasmids were transformed into BL21(DE3) T1^(r) competent cells from Sigmaldrich. Cells were grown in Luria Bertani nutrient medium with 100 μg/mL ampicillin and 0.5 mM IPTG for expression. The codon optimized sequence for eGFP-R₉ is set forth in SEQ ID NO: 5. R₉ as used herein refers to the nonarginine affinity tag. The translated sequence of the eGFP-R₉ is set forth in SEQ ID NO: 6. The amino acids in bold denotes the nonaarginine affinity tag. The “*” of SEQ ID NO: 6 represents the end or “stop” of translation. The “GS” following the “*” represents the amino acids (glycine and serine, respectively) that are included as a translation of the genetic code due to the use of engineered restriction endonuclease sites, but the GS is not part of the translated peptide since the peptide will end coding at the stop codon (“*”).

The sequence for L2 was obtained from the National Center for Biotechnology Information. A fusion protein comprising L2 fused to the C-terminus of SQR was generated. The nucleotide sequence for SQR-L2 is set forth in SEQ ID NO: 7. The translated sequence of SQR-L2 is set forth in SEQ ID NO: 2. The amino acids in bold denotes the L2 affinity tag.

Plasmids were transformed into BL21(DE3) T1^(r) competent cells from Sigma Aldrich. Cells were grown in Luria Bertani nutrient medium with 100 μg/mL ampicillin and 0.5 mM IPTG for expression. Cells were harvested via centrifugation (3,000 RPM for 20 minutes) and the supernatant discarded. Cell pellets were resuspended in dH₂O or Tris Buffered Saline (TB S), pH 7.6 at a ratio of 5 volumes of buffer per 1 volume of cell pellet. Cell debris was removed via centrifugation and the clear supernatant kept as the tagged protein.

Sequence Listings: SEQ ID NO: Sequence  1 MAHVVILGGGTGGMPAAYEMKEALGSGHEVTLISANDYFQFVPSNPW VGVGWKERDDITFPIRHYVERKGIHFVAQSAERIDAEAQNITLADGS TVHYDYLMITAGPKLAFENVPGSDPHEGPVQSICTVDHAERAFAEYQ ALLREPGPIAIGAMAGASCFGPAYEYAMIVASDLKKRGMRDKIPSFT FITSEPYLGHLGIQGVGDSKGILTKGLKEEGIEAYTNCKVTKVEDNK MYVTQVDEKGETIKEMVLPVKLGMMIPAFKGVPAVAGVEGLCNPGGF VLVDEHQRSKKYANIFAAGIAIAIPPVETTPVPTGAPKTGYMIESMV SAAVHNIKADLEGRKGEQTMGTWNAVCFADMGDRGAAFIALPQLKPR KVDVFAYGRWVHLAKVAFEKYFIRKMKIGVSEPFYEKVLFKKMGITR LKEEDAHRKASETHANNAHDAVIDRRRRRRRRR  2 MAHVVILGGGTGGMPAAYEMKEALGSGHEVTLISANDYFQFVPSNPW VGVGWKERDDITFPIRHYVERKGIHFVAQSAERIDAEAQNITLADGS TVHYDYLMITAGPKLAFENVPGSDPHEGPVQSICTVDHAERAFAEYQ ALLREPGPIAIGAMAGASCFGPAYEYAMIVASDLKKRGMRDKIPSFT FITSEPYLGHLGIQGVGDSKGILTKGLKEEGIEAYTNCKVTKVEDNK MYVTQVDEKGETIKEMVLPVKLGMMIPAFKGVPAVAGVEGLCNPGGF VLVDEHQRSKKYANIFAAGIAIAIPPVETTPVPTGAPKTGYMIESMV SAAVHNIKADLEGRKGEQTMGTWNAVCFADMGDRGAAFIALPQLKPR KVDVFAYGRWVHLAKVAFEKYFIRKMKIGVSEPFYEKVLFKKMGITR LKEEDAHRKASETHANNAHAVVKCKPTSPGRRHVVKVVNPELHKGKP FAPLLEKNSKSGGRNNNGRITTRHIGGGHKQAYRIVDFKRNKDGIPA WVVRLEYDPNRSANIALVLYKDGERRYILAPKGLKAGDQIQSGVDAA IKPGNTLPMRNIPVGSTVHNVEMKPGKGGQLARSAGTYVQIVARDGA YVTLRLRSGEMRKVEADCRATLGEVGNAEHMLRVLGKAGAARWRGVR PTVRGTAMNPVDHPGGGHEGRNFGKHPVTPWGVQTKGKKTRSNKRTD KFIVRRRSK  3 RRRRRRRRR  4 AVVKCKPTSPGRRHVVKVVNPELHKGKPFAPLLEKNSKSGGRNNNGR ITTRHIGGGHKQAYRIVDFKRNKDGIPAVVERLEYDPNRSANIALVL YKDGERRYILAPKGLKAGDQIQSGVDAAIKPGNTLPMRNIPVGSTVH NVEMKPGKGGQLARSAGTYVQIVARDGAYVTLRLRSGEMRKVEADCR ATLGEVGNAEHMLRVLGKAGAARWRGVRPTVRGTAMNPVDHPGGGHE GRNFGKHPVTPWGVQTKGKKTRSNKRTDKFIVRRRSK  5 catatggtgagcaaaggcgaagaactgtttaccggcgtggtgccgat tctggtggaactggatg gcgatgtgaacggccataaatttagcgtgagcggcgaaggcgaaggc gatgcgacctatggcaaactgaccctgaaatttatttgcaccaccgg caaactgccggtgccgtggccgaccctggtgaccaccctgacctatg gcgtgcagtgctttagccgctatccggatcatatgaaacagcatgat ttttttaaaagcgcgatgccggaaggctatgtgcaggaacgcaccat tttttttaaagatgatggcaactataaaacccgcgcggaagtgaaat ttgaaggcgataccctggtgaaccgcattgaactgaaaggcattgat tttaaagaagatggcaacattctgggccataaactggaatataacta taacagccataacgtgtatattatggcggataaacagaaaaacggca ttaaagtgaactttaaaattcgccataacattgaagatggcagcgtg cagctggcggatcattatcagcagaacaccccgattggcgatggccc ggtgctgctgccggataaccattatctgagcacccagagcgcgctga gcaaagatccgaacgaaaaacgcgatcatatggtgctgctggaattt gtgaccgcggcgggcattaccctgggcatggatgaactgtataaaga tgatgatgataaagatgcggtgattgatcgccgccgccgccgccgcc gccgccgctgaggatcc  6 HMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGY VQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH KLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNT PIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGM DELYKDDDDKDAVIDRRRRRRRRR*GS  7 catatggcgcatgtggtgattctgggcggcggcaccggcggcatgcc ggcggcgtatgaaatgaaagaagcgctgggcagcggccatgaagtga ccctgattagcgcgaacgattattttcagtttgtgccgagcaacccg tgggtgggcgtgggctggaaagaacgcgatgatattacctttccgat tcgccattatgtggaacgcaaaggcattcattttgtggcgcagagcg cggaacgcattgatgcggaagcgcagaacattaccctggcggatggc agcaccgtgcattatgattatctgatgattaccgcgggcccgaaact ggcgtttgaaaacgtgccgggcagcgatccgcatgaaggcccggtgc agagcatttgcaccgtggatcatgcggaacgcgcgtttgcggaatat caggcgctgctgcgcgaaccgggcccgattgcgattggcgcgatggc gggcgcgagctgctttggcccggcgtatgaatatgcgatgattgtgg cgagcgatctgaaaaaacgcggcatgcgcgataaaattccgagcttt acctttattaccagcgaaccgtatctgggccatctgggcattcaggg cgtgggcgatagcaaaggcattctgaccaaaggcctgaaagaagaag gcattgaagcgtataccaactgcaaagtgaccaaagtggaagataac aaaatgtatgtgacccaggtggatgaaaaaggcgaaaccattaaaga aatggtgctgccggtgaaactgggcatgatgattccggcgtttaaag gcgtgccggcggtggcgggcgtggaaggcctgtgcaacccgggcggc tttgtgctggtggatgaacatcagcgcagcaaaaaatatgcgaacat ttttgcggcgggcattgcgattgcgattccgccggtggaaaccaccc cggtgccgaccggcgcgccgaaaaccggctatatgattgaaagcatg gtgagcgcggcggtgcataacattaaagcggatctggaaggccgcaa aggcgaacagaccatgggcacctggaacgcggtgtgctttgcggata tgggcgatcgcggcgcggcgtttattgcgctgccgcagctgaaaccg cgcaaagtggatgtgtttgcgtatggccgctgggtgcatctggcgaa agtggcgtttgaaaaatattttattcgcaaaatgaaaattggcgtga gcgaaccgttttatgaaaaagtgctgtttaaaatgatgggcattacc cgcctgaaagaagaagatgcgcatcgcaaagcgagcgaaacccatgc gaacaacgcgcatgcggtggtgaaatgcaaaccgaccagcccgggcc gccgccatgtggtgaaagtggtgaacccggaactgcataaaggcaaa ccgtttgcgccgctgctggaaaaaaacagcaaaagcggcggccgcaa caacaacggccgcattaccacccgccatattggcggcggccataaac aggcgtatcgcattgtggattttaaacgcaacaaagatggcattccg gcggtggtggaacgcctggaatatgatccgaaccgcagcgcgaacat tgcgctggtgctgtataaagatggcgaacgccgctatattctggcgc cgaaaggcctgaaagcgggcgatcagattcagagcggcgtggatgcg gcgattaaaccgggcaacaccctgccgatgcgcaacattccggtggg cagcaccgtgcataacgtggaaatgaaaccgggcaaaggcggccagc tggcgcgcagcgcgggcacctatgtgcagattgtggcgcgcgatggc gcgtatgtgaccctgcgcctgcgcagcggcgaaatgcgcaaagtgga agcggattgccgcgcgaccctgggcgaagtgggcaacgcggaacata tgctgcgcgtgctgggcaaagcgggcgcggcgcgctggcgcggcgtg cgcccgaccgtgcgcggcaccgcgatgaacccggtggatcatccggg cggcggccatgaaggccgcaactttggcaaacatccggtgaccccgt ggggcgtgcagaccaaaggcaaaaaaacccgcagcaacaaacgcacc gataaatttattgtgcgccgccgcagcaaatgaggatcc  8 MAHVVILGAGTGGMPAAYEMKEALGSGHEVTLISANDYFQFVPSNPW VGVGWTKRDDIAFPIKPYVERKGIHFIPKAAEKIDAEGQEITLADGS KVRYDYLLITTGPKLAFENVPGSDPHEGPIQSICTVDHAEKAYHDYQ ALLAEPGPIVIGAMGGASCFGPAYEYAMVVASDLKKRGMRDKISSFT FVTSEPYLGHLGIQGVGDSTGILSKGLKEEGIEAYTNCKVTKVEGGK MFVTQVNDKGEVAKEFTLPVKFGMMIPAFKGVPAVAGVEGLCNPGGF VLVDEHQRSKKYANIFAAGIAIAIPPVEATPVPTGAPKTGYMIESMV SAAVHNIKADLEGRKGEQTMGTWNAVCFADMGDRGAAFVALPQLRPR KVDVFAYGRWVHLAKVAFEKYFIRKMKMGVSEPFYEKVLFKKMGITR LKEEVPPHRKAS  9 MAHIVILGAGTGGTPAAYEMREALGREHKVTLINASETFQFVPSNPW VAVGWRERDDTTFPLRQYVEKKGINFIADRVDRIDPEANQLTLAGGD TVDYDYLVLTTGPMLAFDEVEGTGPHDGYTQSVCTIDHAETAYQKYE EFLKNPGPVVVGAVQGASCFGPAYEFAMILDRDLRKRKMRDQVPITF VTSEPYIGHMGLGGVGDSKGLLEHELRERHINWITNARTERVEDGKM YVTQLDEKGEVLKEHELDFNYSMMLPAFRGVPAVADVEGLCNPRGFV KVDECQRSPAYSNIFAAGVGIAIPPVEQTTVATGAPKTGYMIESMVT AIVENIAGEVEGKGGCNTQGTWSTICLADLGDTGAAFVALPQIPPRN VTWSKKGKWVHYAKIAFEKYFMRKMKTGHSEPIYEKYVLRMLGINRL KK 10 MAHVVVLGAGTGGMPCAYELRAELGREHEVTMINEREYFQFVPSNPW LAVGWRDRSHITFDIRPHLERKGINFIAKRVDKIDAEGNKLELDDGE TIEYDYLVIATGPRLAFEEVEGSGPEGHTQSICTVNHAEKAFDAYKD LLDEPGPVIIGAMPFASCFGPAYEFSFIMDSDLRKRKMRDKVPMTYV TSEPYIGHLGLGGVGDSKGFLESDFRAHHINWITNAKVIKVEAGKMF VEQYDDSGHKIKEHELEFKYSMMLPAFKGVDAVANVEGLCNPRGFVF VDDHQCNPTYKNIYAAGVCIAIPPVEATAVPTGAPKTGYMIESMVTA IVHNIADDLAGKEGTTLATWNAICLADMGDTGAAFVALPQIPPRNVA WFKKGKWVHMAKIAFEKYFIRKMKKGTSEPIYEKYILKMLGIGKLK 11 MAHIVVIGAGIGGMPAAYELRSKLPAQHRVTVISAVDYFHFVPSNPW IAVGWRQREDIVLQLAPLLQRKGIDFIASPVQTIDAAGNSLALANGQ TVAYDYLVITTGPRLAFEEVPGAGPIDGHTHSICTVDHAQHFWADYE KFLENPGPMVIGAMPGASCFGPAYEFAFIVSADLRKRKLRHKVPLTY VTSEPYIGHLGLGGVGDSKSMLESELRGQDIKWITNAKTTRVEDGKM MVDQLDDQGKLLKQHELPFKLSMMLPAFKGVDAVAAVPSLCNPRGFV LIDAHQRSKAYPNIFAAGVCVAIPPVEVTPVPTGAPKTGYMIETMVS AIVHNIAADLEGKPATATATWNAICLADMGDTGAAFVALPQIPPRNV NWFKKGKWVHLGKIAFEKYFLGKIKSGNTDPIYEKYVLKILGIERLP EPSGPR 12 MAHIVILGAGTGGMPAAYEMKEMLGKGHEVTVVNERDYFQFVPSNPW VAVGWRTRSDITFPIEKYLSKKDIKFICSRCEKIDAEGNALHLADGQ IVKYDYLVIATGPKLFFQEVEGAGPHGGHTHSVCDVTHAEGAYADYQ KLLAKGSGHIIVGAMPFASCFGPAYEFAFIVDADLRKRGLRHKFKMT YVSSEPYIGHLGLGGVGDSKGMLESELRNHHMGWITNAKTTKVEAGK MHVTEMTAKGEVEKEHVIDFDMAMMLPAFKGVDAVAAVEGLCNPRGF VIVDELHRSPKYKNIYSAGVCIAIPPVEATPVPTGAPKTGYMIESMV TSLVHNIADELAGKEPHTTATWNAICLADMGDTGAAFVALPQIPPRN VAWFKKGKWVHMAKIAFEKYFIRKMKKGSSEPFYEKSILKM:MGITRI

eGFP-R₉ proteins were mixed with sand, sandstone wafers, glass beads or glass slides and visualized in a dark room with ultraviolet light (360 nm). A green emission indicates the presence of GFP. The tagged protein contacted the substrate at 37° C. for at least an hour. Samples were then removed from the protein fluid and washed with at least 10 volumes of dH2O and then visualized at 360 nM. As shown in FIG. 1, the tagged eGFP fluoresces brightly. The sample vials on the left and in the middle have eGFP-R₉ tagged sand. The sand in the tube on the left has been washed with 50 sample volumes of dH₂O. The tube in the middle has not been washed. The tube on the left fluoresces under these conditions indicating the presence of GFP. The tube on the right contains moist sand without eGFP.

Referring to FIG. 2, the image at the top shows the sandstone wafers in natural light. The image at the bottom was taken in ultraviolet light (360 nM). The sandstone wafer on the left was stained for 1 hour at room temperature with eGFP-R9 and then washed with 100 sample volumes of distilled water (dH2O). The wafer on the right was saturated with dH2O only. This indicates that the tag was effective in binding the protein to the wafer. Interestingly, this wafer still fluoresced after drying. Even after 4 weeks of being dry, this wafer still displayed a strong fluorescent signal.

Referring to FIG. 3, the experiment was similar to FIG. 2 except that sandstone cores were used. The treated sample (left) fluoresces strongly in the presence of ultraviolet light. This sandstone core was exposed to eGFP-R₉ for 1 hour at room temperature and then washed with 80 core volumes of dH₂O.

The eGFP-R₉ fusion produced the most conclusive results in these tests. In contrast, it was difficult to produce results with the SQR-L2 enzyme system because the sulfide in solution readily reacted with the sand, wafers and proppant. For example, tests routinely produced false negatives (and sometimes positives) as it was difficult to get a reproducible result with sulfides and solid supports. On average, there were fewer sulfides detected in enzyme treated samples than blank samples, indicating that the enzyme was present and active. We further performed experiments in aqueous solution without the solid substrate present. The goal of the test was to determine if the presence of the 30 KDa tag abolished the enzyme's activity. SQR-L2 was suspended in Tris buffered Saline, pH7.6 and the applied to solutions containing 200 ppm sodium sulfide. Enzymes were allowed to react with the sulfide for 2 hours at room temperature and then the sulfide concentration measured with the HACH HS-C filter paper method from HACH, Colorado. A stock solution of 200 ppm sodium sulfide was used as a sulfide source. A black test paper indicated the presence of sulfide. Therefore, the presence of the active SQR enzyme was shown by an unreacted test paper. As an alternative test, the HACH sulfide test kit was used. The same 200 ppm stock solution of sodium sulfide was used and the presence of sulfide was indicated by the formation of methylene blue (blue color) in liquid samples. Quantification of enzyme activity could be measured by using absorbance at 650 nm. Enzyme treated samples contained 2 mg/L sulfide while the untreated sample was measured off the charts at ≥5 mg/L of sulfide. This indicated that the enzyme was present and functional and that the affinity tag did not abolish the activity of the enzyme.

The R₉ and L2 tags do not abolish the activity of the enzymes and the R₉ tag clearly adheres to the silicate surfaces as evidenced by the strong fluorescence reporting of the treated samples. Even when the samples are washed, the protein remains affixed to the silicate surfaces. While it was more difficult to measure the efficacy of the SQR-L2 fusion, results clearly indicated that the enzyme was present and functional.

Publications cited throughout this document are hereby incorporated by reference in their entirety. Although the various aspects of the invention have been illustrated above by reference to examples and preferred embodiments, it will be appreciated that the scope of the invention is defined not by the foregoing description but by the following claims properly construed under principles of patent law. 

What is claimed is:
 1. A recombinant protein, comprising: an affinity tag configured to attach the recombinant protein to a silicate surface, fused to a hydrogen sulfide scavenging enzyme, wherein the recombinant protein comprises the protein having the amino acid sequence set forth in SEQ ID NO:
 1. 2. A recombinant protein, comprising: an affinity tag configured to attach the recombinant protein to a silicate surface, fused to a hydrogen sulfide scavenging enzyme, wherein the recombinant protein comprises the protein having the amino acid sequence set forth in SEQ ID NO:
 2. 